Effectiveness of Plasmocure™ in Elimination of Mycoplasma Species from Contaminated Cell Cultures: A Comparative Study versus Other Antibiotics

Objective Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problems associated with cell culture in biology research centers or biotechnological companies. Mycoplasmas are also resistant to several antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported. InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasma contamination. The aim of this study was the selection of the best antibiotics for treatment of mycoplasma in cell cultures. Materials and Methods In this experimental study, a total of 100 different mammalian cell lines contaminated with different Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochrome staining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods. In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. The treatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BM- cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. Results Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those in Plasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma spp. A moderate toxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells were fully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. However, the outcome of the treatment process (i.e. the frequency of complete cure, regrowth or cell death) varied among different antibiotics. Conclusion The highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowest regrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.


Introduction
Mycoplasma spp. contaminations cause a wide range of economical and biotechnical troubles in cell cultures in biological research laboratories as well as biotechnology companies (1,2). In 1956, Mycoplasma was described as one of the most important contaminants of cell cultures (3). Most of the Mycoplasma species are known as saprophytic and commensal microbes in eukaryotes (4,5). They are the smallest and simplest self-replicating bacteria lacking cell wall properties. The cell membrane of Mycoplasma is made of triple-layers of cholesterol.  (6). Mycoplasma contaminations can affect the proliferation, the morphology, as well as the metabolic properties of the infected cells. Mycoplasma infections may also alter the genome, transcriptome, and proteome properties of the host cells and alter their plasma membrane antigens (1,4).
Methods for eliminating Mycoplasmas from cell cultures include physical, chemical, immunological, and antibiotic-based approaches. Nevertheless, the methods of Mycoplasma elimination should ideally be simple, rapid, efficient, reliable, and inexpensive. They should also have minimal effects on cultured eukaryotic cells (7,8). Three groups of antibiotics namely, tetracyclines, macrolides and fluoroquinolones, have been shown to be highly effective against Mycoplasmas in patients or in cell culture. Since each antibiotic has a specific activity and might not completely eliminate all the Mycoplasmas present in a culture, using a combination Plasmocure™ for Elimination of Mycoplasma of antibiotics has been frequently implemented (9,10). The InvivoGen Company has introduced several antibiotics with different mechanisms of action to treat Mycoplasma-contaminated cell cultures. In particular, Plasmocin ™ (InvivoGen, USA, Cat No. ant-mpt version 16F09-MM) is used to treat cell lines infected by Mycoplasmas and related cell wall-less bacteria. Plasmocin ™ can also be used as prophylaxis for Mycoplasma and other bacterial contaminations. However, some Mycoplasmas have been reported to be resistant to Plasmocin ™ (8,11). To eradicate these Mycoplasmas, InvivoGen has developed a new antimycoplasma agent called Plasmocure ™ (Alternative Mycoplasma Removal Agent, InvivoGen, USA, Cat No. ant-pc version 16F09-MM). Plasmocure ™ is comprised of two antibiotics that act through mechanisms different from those of Plasmocin ™ . Two-week treatment with Plasmocure ™ is enough to completely eradicate Mycoplasmas (12). In the present study, we aimed to compare the efficacy and cytotoxicity of Plasmocure ™ versus five other available antibiotics namely, Plasmocin ™ , BM-cyclin (Roche), MycoRAZOR ™ , sparfloxacin and enrofloxacin. To this end, we evaluated the effectiveness of these antibiotics in elimination of different Mycoplasma species contaminating various mammalian cell lines, at National Cell Bank of Iran (NCBI).
The working concentrations of Plasmocure™, BMcyclin (Roche), Plasmocin™ and MycoRAZOR™ were chosen according to the manufacturer's instructions. Furthermore, sparfloxacin and enrofloxacin working concentrations were determined according to previously published reports (7,12,(16)(17)(18). Following treatment with these reagents, the cells were cultured without penicillin, streptomycin or other commonly-used antibiotics (i.e. under antibiotic-free conditions) for at least another 1-2 weeks prior to testing for residual Mycoplasma contamination. All the cured cultures were re-examined for regrowth of Mycoplasmas for 4 months following the treatment (10).

Detection of Mycoplasma contamination by microbiological culture
The suspended cells (1 ml) were added to 10 ml of Pleuropneumonia-Like Organisms (PPLO) broth medium (BD Difco ™ , USA) supplemented with 10% horse serum (Gibco ® , New Zealand), 1% yeast extract agar (Sigma-Aldrich ® , Germany), L-arginine (Sigma-Aldrich ® , Germany), D-glucose (Dextrose, Gibco ® , USA) and cultured at 37˚C for 48-72 hours. In the next step, PPLO medium was vigorously stirred to observe monotonous turbidity. After centrifugation at 1500 rpm for 15 minutes, the precipitate (100 μl) was transferred to a solid PPLO agar (BD Difco ™ , USA) culture plate and incubated at 37˚C for 4-6 weeks. Microscopy observation was used to investigate the formation of non-typical colonies or egg form of Mycoplasma colonies, every 3-4 days (1).

Detection of Mycoplasma contamination by indirect DNA DAPI staining
This experiment was performed according to previous published reports (21,22). Briefly, cells were cultured on cover slips and stained with 4´, 6-diamidine-2´-phenylindoledihydrochloride (DAPI, Roche, Germany) working solution in methanol (1 μg/ml) at 37˚C for 15 minutes. Mycoplasma bodies were detected as polymorphous particles with blue fluorescence. For indirect staining, the supernatants of cell cultures that were suspected to be contaminated with Mycoplasma, were added to the Mycoplasma-free Vero cell line (NCBI C101b, National Cell Bank of Iran) (19,20).

Detection of Mycoplasma contamination by MycoAlert ™ Mycoplasma detection kit
The enzymatic MycoAlert ™ Mycoplasma detection kit was used according to the manufacturer's instruction. Briefly, the ratio of the ATPs level in each sample before (Reading A) and after (Reading B) the addition of MycoAlert ™ substrate, was considered an indicator for the presence of Mycoplasma contamination. The presence of contamination was proved if the Reading B/Reading A ratio was greater than 1 (1,21,22).

Mycoplasma detection using universal and specific polymerase chain reaction method
The Mycoplasma contamination status in 100 cell lines (Table S1) (See Supplementary Online Information at www. celljournal.org) was also determined using PCR-based method as described previously (9,20). In addition to universal primer pair, 11 species-specific primer pairs were designed based on the 16SrRNA of mollicutes (Fig.1). Sequences of all primers were previously published (20,21).

Determination of Mycoplasma contamination status in control cell lines
The control cell lines of the study were assessed for Mycoplasma contamination using microbiological culture (as the reference standard test), indirect DNA DAPI staining, enzymatic MycoAlert ™ and PCR detection (with universal and specific primers) methods. Vero cell line (NCBI C101a) contaminated with several Mycoplasma species, and

Statistical analysis
Statistical analysis was performed using SPSS 24.0 software (IBM ® SPSS ® Statistics, USA). Non-parametric Chi-square test (χ2) was used for comparisons of two-bytwo in six groups. In Chi-square tests, the difference among the antibiotics for the treatment of Mycoplasma-infected cell lines was analyzed and interpreted. Differences with a P<0.05 were considered statistically significant.

Characteristics, frequency and treatment of Mycoplasma contaminations
In this study, 100 different human and animal cell lines were randomly selected and assessed for mycoplasma contamination. The type of mollicutes in each cell line determined by PCR-based method indicating that 65/100 (65%) of the infected cell cultures was contaminated by one Mycoplasma species. Moreover, 19/100 (19%) samples were contaminated with two species and 16/100 (16%) were contaminated with three different species (Table S1)

Eradication of Mycoplasma contaminations
The results obtained from Mycoplasma treatment process are summarized in Table S1 (See Supplementary Online Information at www.celljournal.org) and Figure 2. Mycoplasma infections were eliminated by Plasmocure ™ , BM-cyclin (Roche), Plasmocin ™ , MycoRAZOR ™ ,sparfloxacin and enrofloxacin in 91, 70, 66, 55, 33 and 15% of the contaminated cell cultures, respectively. Furthermore, decontamination was confirmed by PCR, as no Mycoplasma was detected in cured cell cultures 14 days after the completion of the treatment period. Mycoplasma regrowth (reinfection or recurrent infection) was observed in 3, 12, 17, 42, 62 and 83% of the cured cell lines four months after treatment with Plasmocure™, Plasmocin™, BMcyclin (Roche), MycoRAZOR ™ , sparfloxacin and enrofloxacin, respectively. According to the obtained results, the highest level (22%) of cell cytotoxicity (culture death) was observed among Plasmocin-treated cell lines. While, BM-cyclin (Roche), Plasmocure ™ , sparfloxacin, MycoRAZOR ™ andenrofloxacin were cytotoxic to up to 13, 6, 5, 3 and 2% of the studied cell lines, respectively (Table S1 [See Supplementary Online Information at www.celljournal.org], Fig.2). The outcome in the 6 groups of antibiotics showed a significant difference in two-by-two comparison antibiotics in reciprocal case ( Table 2). There were significant differences between Plasmocure ™ and other antibiotics (P=0.001) with regard to treatment of contaminated cell cultures ( Table 2, Fig.2).
However, there was no significant difference between Plasmocin ™ and BM-cyclin in the comparison of the treatment outcome (P=0.193). Overall, results reported on antibiotic treatments of cell cultures by different studies are summarized in Table 3.

Discussion
Mycoplasma contamination remains one of the major problems in cell culture laboratories. Mycoplasmas can cause significant biological changes in cultured mammalian cells. In fact, consequences of Mycoplasma contamination are unpredictable and may affect molecular and cellular properties of the infected cells (7,23,24). In particular, Mycoplasma contamination can lead to attenuation of cell proliferation, unreliable experimental results, and potentially unsafe biological products (1,25). Mycoplasmas are resistant to many antibiotics which are commonly used in cell culture. This problem Plasmocure™ for Elimination of Mycoplasma has become more widespread since the introduction of more sensitive, rapid, and efficient methods of detection of Mycoplasmas in cell culture. Recent reports have estimated that Mycoplasma contamination may affect up to 83% of cell cultures worldwide (2,4,6,10). Administration of antibiotics is the most reliable and efficient approach to combat Mycoplasma contamination. However, it is important to determine the efficacy and potential side-effects of the antibiotics on the eukaryotic cells in culture. For treatment of irreplaceable, valuable and expensive cell lines, the safety of the antibiotics used against Mycoplasma contamination, is particularly important (2,4,8). In addition, some cell types may be infected with different Mycoplasma species making it difficult to draw an accurate conclusion on choosing an antibiotic (7,26,27). In our experience, Plasmocure ™ was able to cure 91 out of 100 cell lines (91%), with 3 cases of regrowth (3%) and 6 of cell death (6%). Plasmocure ™ is comprised of two bactericidal components belonging to different antibiotic families. They both act by inhibiting protein synthesis but through distinct mechanisms. One of these antibiotic binds to the 50s subunit of the bacterial ribosomeand blocks the peptidyltransferase activity. The other antibiotic which binds to isoleucyl-tRNAsynthetase prevents the addition of isoleucine to bacterial proteins (28)(29)(30).
Remarkably, the problem of regrowth in Plasmocure ™treated cell lines was resolved by using Plasmocin ™ or BMcyclin (Roche), and vice versa. In case of cytotoxicity and cell death, especially in severe and intensive contaminations with multiple Mycoplasma strains, BM-cyclin (Roche) and Plasmocin ™ were used successfully. In case of mild contaminations, particularly for vulnerable or precious cells such as myeloma, lymphoma, hybridoma or primary cultures, MycoRAZOR ™ along with fluoroquinolones (sparfloxacin and enrofloxacin) can be used as alternative antibiotics. Plasmocin ™ (comprised of a macrolide and a quinolone) acts on the protein machinery and DNA replication by interfering with ribosomal translation and replication fork, respectively. BM-cyclin (Roche) binds to the 30S and 50S ribosomal subunits and inhibits protein synthesis. According to the manufacturer's information, the bacteriostatic components of BM-cyclin (Roche) are pleuromutilin and tetracycline whereas Plasmocin ™ is composed of a macrolide and a quinolone (10-12, 31, 32). MycoRAZOR ™ is an effective antibiotic against Mycoplasma, which is active at low concentrations against various Mycoplasma species. It diminishes Mycoplasmas protein biosynthesis by interfering with their ribosome function as well as DNA transcription. MycoRAZOR ™ has no undesired impact on the eukaryotic cells in the culture. On the other hand, sparfloxacin and enrofloxacin as members of the fluoroquinolone family, inhibit bacterial DNA gyrase and DNA replication (33).
In this study, we observed high frequency of Mycoplasma resistance/regrowth following treatment with enrofloxacin (83%), sparfloxacin (62%) or MycoRAZOR ™ (42%). Plasmocure ™ , BM-cyclin (Roche) and Plasmocin ™ were effective especially in elimination of M.hyorhinis, M.arginini, M.fermentans and M.orale which were resistant to the other antibiotics used in this study. Plasmocure™ showed the lowest frequency (3%) of regrowth in our experiments while regrowth was observed in 17 and 12% of cell lines treated with BM-cyclin (Roche) and Plasmocin ™ , respectively. Plasmocure ™ , BM-cyclin (Roche), Plasmocin ™ and MycoRAZOR ™ effectively eradicated mollicutes and cured 91, 70, 66 and 55% of the cell lines, respectively. However, sparfloxacin and enrofloxacin were considerably less efficient as they cured only 33 and 15% of the cell lines, respectively. Plasmocin ™ caused the highest rate of culture death (22%), although, it targets the prokaryotic DNA replication and protein synthesis machineries which are different from those of eukaryotic cells. In addition, MycoRAZOR ™ showed lower cytotoxicity on the studied cell lines (culture death of 3%), which might reduce the risk of culture loss. Therefore, it may be recommended as the first-line alternative especially in case of expensive or hard-to-obtain cell lines (9,10,34). Moreover, quinolones and fluoroquinolones such as ciprofloxacin, enrofloxacin or sparfloxacin were used along with MycoRAZOR ™ without increased cytotoxicity (35).
Finally, we observed that the combination of two or more antibiotic with different mechanisms of action makes the interpretation of the results more complicated. Based on our experiments, this might increase the risk of culture death or antibiotic resistance of Mycoplasmas. Thus, we suggest using two or more antibiotics in alternating periods for a successful treatment or eradication of Mycoplasma contamination. For example, BM-cyclin (Roche) or MycoRAZOR ™ (inhibitors of protein synthesis) can be used alternately along with ciprofoxacin, enrofloxacin or sparfloxacin (inhibitors of DNA gyrase activity and DNA replication) with specified intervals during the treatment period.

Conclusion
This report suggests Plasmocure ™ as a reliable anti-Mycoplasma agent in comparison with other antibiotics for elimination of Mycoplasma contamination in cultured cells. As a conclusion, we recommend Plasmocure ™ as an effective antibiotic for the treatment of Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells. These findings may also help researches at biotechnology laboratories for selection of appropriate antibiotics for treatment of Mycoplasma contamination in cell cultures.